The S49 Kin- cell line transcribes and translates a functional mRNA coding for the catalytic subunit of cAMP-dependent protein kinase.
The S49 mouse lymphoma mutant cell line Kin- is resistant to the cytotoxic effects of elevated cAMP levels, has no detectable cAMP-dependent protein kinase activity, and has depressed levels of cAMP-binding regulatory subunits. We demonstrate that although the Kin- cell line lacks detectable catalytic subunit protein, these cells express wild-type levels of mRNA for both C alpha and C beta catalytic subunit isoforms. Translation of C alpha mRNA appears to be normal in the Kin- cell, based on the observation that C alpha mRNA associates with large polyribosomes in both wild-type and Kin- cells. We cloned the C alpha cDNA from Kin- cells and show that its transient expression in another cell type leads to activation of a cAMP-sensitive luciferase reporter gene, suggesting that functional C alpha protein is made. In addition to having catalytic activity, the C alpha subunit from Kin- cells is inhibited in the presence of mouse RI alpha regulatory subunit, indicating that formation of the holoenzyme complex is normal. We suggest that the mutation responsible for the Kin- phenotype is in a cellular component that directly or indirectly causes Kin- catalytic subunit protein to be degraded rapidly.
Orellana
S A
SA
Department of Pharmacology, University of Washington, Seattle 98195.
McKnight
G S
GS
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D008223:Lymphoma / Q000201:enzymology / Q000235:genetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011494:Protein Kinases